T7 DNA polymerase

ABSTRACT

This invention relates to T7-type DNA polymerases and method for using them.

BACKGROUND OF THE INVENTION

This invention was made with government support including a grant fromthe U.S. Public Health Service, contract number AI-06045. The governmenthas certain rights in the invention.

This invention relates to DNA polymerases suitable for DNA sequencing.

DNA sequencing involves the generation of four populations of singlestranded DNA fragments having one defined terminus and one variableterminus. The variable terminus always terminates at a specific givennucleotide base (either guanine (G), adenine (A), thymine (T), orcytosine (C)). The four different sets of fragments are each separatedon the basis of their length, on a high resolution polyacrylamide gel;each band on the gel corresponds colinearly to a specific nucleotide inthe DNA sequence, thus identifying the positions in the sequence of thegiven nucleotide base.

Generally there are two methods of DNA sequencing. One method (Maxam andGilbert sequencing) involves the chemical degradation of isolated DNAfragments, each labeled with a single radiolabel at its definedterminus, each reaction yielding a limited cleavage specifically at oneor more of the four bases (G, A, T or C). The other method (dideoxysequencing) involves the enzymatic synthesis of a DNA strand. Fourseparate syntheses are run, each reaction being caused to terminate at aspecific base (G, A, T or C) via incorporation of the appropriate chainterminating dideoxynucleotide. The latter method is preferred since theDNA fragments are uniformly labelled (instead of end labelled) and thusthe larger DNA fragments contain increasingly more radioactivity.Further, ³⁵ S-labelled nucleotides can be used in place of ³² P-labellednucleotides, resulting in sharper definition; and the reaction productsare simple to interpret since each lane corresponds only to either G, A,T or C. The enzyme used for most dideoxy sequencing is the Escherichiacoli DNA-polymerase I large fragment ("Klenow"). Another polymerase usedis AMV reverse transcriptase.

SUMMARY OF THE INVENTION

In one aspect the invention features a method for determining thenucleotide base sequence of a DNA molecule, comprising annealing the DNAmolecule with a primer molecule able to hybridize to the DNA molecule;incubating separate portions of the annealed mixture in at least fourvessels with four different deoxynucleoside triphosphates, a processiveDNA polymerase having less than 500 units of exonuclease activity per mgof polymerase, and a DNA synthesis terminating agent which terminatesDNA synthesis at a specific nucleotide base. The agent terminates at adifferent specific nucleotide base in each of the four vessels. The DNAproducts of the incubating reaction are separated according to theirsize so that at least a part of the nucleotide base sequence of the DNAmolecule can be determined.

In preferred embodiments the polymerase remains bound to the DNAmolecule for at least 500 bases before dissociating, most preferably forat least 1,000 bases; the polymerase is substantially the same as one incells infected with a T7-type phage (i.e., phage in which the DNApolymerase requires host thioredoxin as a subunit) for example, theT7-type phage is T7, T3, ΦI, ΦII, H, W31, gh-1, Y, A1122, or Sp6; thepolymerase is non-discriminating for dideoxy nucleotide analogs; thepolymerase is modified to have less than 50 units of exonucleaseactivity per mg of polymerase, more preferably less than 1 unit, evenmore preferably less than 0.1 unit, and most preferably has nodetectable exonuclease activity; the polymerase is able to utilizeprimers of as short as 10 bases or preferably as short as 4 bases; theprimer comprises four to forty nucleotide bases, and is single strandedDNA or RNA; the annealing step comprises heating the DNA molecule andthe primer to above 65 ° C., preferably from 65° C. to 100° C., andallowing the heated mixture to cool to below 65° C., preferably to 10°C. to 30° C.; the incubating step comprises a pulse and a chase step,wherein the pulse step comprises mixing the annealed mixture with allfour different deoxynucleoside triphosphates and a processive DNApolymerase, wherein at least one of the deoxynucleoside triphosphates islabelled; most preferably the pulse step performed under conditions inwhich the polymerase does not exhibit its processivity and is for 30seconds to 20 minutes at 0° C. to 20° C. or where at least one of thenucleotide triphosphates is limiting; and the chase step comprisesadding one of the chain terminating agents to four separate aliquots ofthe mixture after the pulse step; preferably the chase step is for 1 to60 minutes at 30° C. to 50° C.; the terminating agent is adideoxynucleotide, or a limiting level of one deoxynucleosidetriphosphate; one of the four deoxynucleotides is chosen from dITP ordeazaguanosine; and labelled primers are used so that no pulse step isrequired, preferably the label is radioactive fluorescent.

In other aspects the invention features (a) a method for producing bluntended double-stranded DNA molecules from a linear DNA molecule having no3' protruding termini, using a processive DNA polymerase free fromexonuclease activity; (b) a method of amplification of a DNA sequencecomprising annealing a first and second primer to opposite strands of adouble stranded DNA sequence and incubating the annealed mixture with aprocessive DNA polymerase having less than 500 units of exonuceaseactivity per mg of polymerase, preferably less than 1 unit, wherein thefirst and second primers anneal to opposite strands of the DNA sequence;in preferred embodiments the primers have their 3' ends directed towardeach other; and the method further comprises, after the incubation step,denaturing the resulting DNA, annealing the first and second primers tothe resulting DNA and incubating the annealed mixture with thepolymerase; preferably the cycle of denaturing, annealing and incubatingis repeated from 10 to 40 times; (c) a method for in vitro mutagenesisof cloned DNA fragments, comprising providing a cloned fragment andsynthesizing a DNA strand using a processive DNA polymerase having lessthan 1 unit of exonuclease activity per mg of polymerase; (d) a methodof producing active T7-type DNA polymerase from cloned DNA fragmentsunder the control of non-leaky promoters (see below) in the same cellcomprising inducing expression of the genes only when the cells are inlogarithmic growth phase, or stationary phase, and isolating thepolymerase from the cell; preferably the cloned fragments are under thecontrol of a promoter requiring T7 RNA polymerase for expression; (e) agene encoding a T7-type DNA polymerase, the gene being geneticallymodified to reduce the activity of naturally occurring exonucleaseactivity; (f) the product of the gene encoding genetically modifiedpolymerase; (g) a method of purifying T7 DNA polymerase from cellscomprising a vector from which the polymerase is expressed, comprisingthe steps of lysing the cells, and passing the polymerase over a sizingcolumn over a DE52 DEAE column, a phosphocellulose column, and ahydroxyapatite column; preferably prior to the passing step the methodcomprises precipitating the polymerase with ammonium sulfate; the methodfurther comprises the step of passing the polymerase over a sephadexDEAE50 column; and the sizing column is a DE52 DEAE column; (h) a methodof inactivating exonuclease activity in a DNA polymerase solutioncomprising incubating the solution in a vessel containing oxygen, areducing agent and a transition metal; (i) a kit for DNA sequencing,comprising a processive DNA polymerase having less than 500 units ofexonuclease activity per mg of polymerase, wherein the polymerase isable to exhibit it processivity in a first environmental condition, andunable to exhibit its processivity in a second environmental condition,and a reagent necessary for the sequencing, selected from adeoxynucleotide, a chain terminating agent, or an oligonucleotideprimer; preferably the deoxynucleotide is dITP; (j) a method forlabelling the 3' end of a DNA fragment comprising incubating the DNAfragment with a processive DNA polymerase having less than 500 units ofexonuclease activity per mg of polymerase, and a labelleddeoxynucleotide; (k) a method for in vitro mutagenesis of a cloned DNAfragment comprising providing a primer and a template, the primer andthe template having a specific mismatched base, and extending the primerwith a processive DNA polymerase; and (l) a method for in vitromutagenesis of a cloned DNA fragment comprising providing the clonedfragment and synthesizing a DNA strand using a processive DNApolymerase, having less than 50 units of exonuclease activity, underconditions which cause misincorporation of a nucleotide base.

This invention provides a DNA polymerase which is processive,non-discriminating, and can utilize short primers. Further, thepolymerase has no associated exonuclease activity. These are idealproperties for the above described methods, and in particular for DNAsequencing reactions, since the background level of radioactivity in thepolyacylamide gels is negligible, there are few or no artifactual bands,and the bands are sharp--making the DNA sequence easy to read. Further,such a polymerase allows novel methods of sequencing long DNA fragments,as is described in detail below.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof and from theclaims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The drawings will first briefly be described.

DRAWINGS

FIGS. 1-3 are diagrammatic representations of the vectors pTrx-2,mGP1-1, and pGP5-5 respectively;

FIG. 4 is a graphical representation of the selective oxidation of T7DNA polymerase;

FIG. 5 is a graphical representation of the ability of modified T7polymerase to synthesize DNA in the presence of etheno-dATP; and

FIG. 6 is a diagrammatic representation of the enzymatic amplificationof genomic DNA using modified T7 DNA polymerase.

FIGS. 7, 8 and 9 are the nucleotide sequences of pTrx-2, a part ofpGP5-5 and mGP1-2 respectively.

DNA Polymerase

In general the DNA polymerase of this invention is processive, has noassociated exonuclease activity, does not discriminate againstnucleotide analog incorporation, and can utilize small oligonucleotides(such as tetramers, hexamers and octamers) as specific primers. Theseproperties will now be discussed in detail.

Processivity

By processivity is meant that the DNA polymerase is able to continuouslyincorporate many nucleotides using the same primer-template withoutdissociating from the template. The degree of processivity varies withdifferent polymerases: some incorporate only a few bases beforedissociating (e.g. Klenow, T4 DNA polymerase, and reverse transcriptase)while others, such as those of the present invention, will remain boundfor at least 500 bases and preferably at least 1,000 bases undersuitable environmental conditions. Such environmental conditions includehaving adequate supplies of all four deoxynucleoside triphosphates andan incubation temperature from 10° C.-50° C. Processivity is greatlyenhanced in the presence of E. coli single stranded binding (ssb),protein.

With processive enzymes termination of a sequencing reaction will occuronly at those bases which have incorporated a chain terminating agent,such as a dideoxynucleotide. If the DNA polymerase is non-processive,then artifactual bands will arise during sequencing reactions, atpositions corresponding to the nucleotide where the polymerasedissociated. Frequent dissociation creates a background of bands atincorrect positions and obscures the true DNA sequence. This problem ispartially corrected by incubating the reaction mixture for a long time(30-60 min) with a high concentration of substrates, which "chase" theartifactual bands up to a high molecular weight at the top of the gel,away from the region where the DNA sequence is read. This is not anideal solution since a non-processive DNA polymerase has a highprobability of dissociating from the template at regions of compactsecondary structure, or hairpins. Reinitiation of primer elongation atthese sites is inefficient and the usual result is the formation ofbands at the same position for all four nucleotides, thus obscuring theDNA sequence.

Analog discrimation

The DNA polymerases of this invention do not discriminate significantlybetween dideoxy-nucleotide analogs and normal nucleotides. That is, thechance of incorporation of an analog is approximately the same as thatof a normal nucleotide. The polymerases of this invention also do notdiscriminate significantly against some other analogs. This is importantsince, in addition to the four normal deoxynucleoside triphosphates(dGTP, dATP, dTTP and dCTP), sequencing reactions require theincorporation of other types of nucleotide derivatives such as:radioactively- or fluorescently-labelled nucleoside triphosphates,usually for labeling the synthesized strands with ³⁵ S, ³² P, or otherchemical agents. When a DNA polymerase does not discriminate againstanalogs the same probability will exist for the incorporation of ananalog as for a normal nucleotide. For labelled nucleoside triphosphatesthis is important in order to efficiently label the synthesized DNAstrands using a minimum of radioactivity. Further, lower levels ofanalogs are required with such enzymes, making the sequencing reactioncheaper than with a discriminating enzyme.

Discriminating polymerases show a different extent of discriminationwhen they are polymerizing in a processive mode versus when stalled,struggling to synthesize through a secondary structure impediment. Atsuch impediments there will be a variability in the intensity ofdifferent radioactive bands on the gel, which may obscure the sequence.

Exonuclease Activity

The DNA polymerase of the invention has less than 50%, preferably lessthan 1%, and most preferably less than 0.1%, of the normal or naturallyassociated level of exonuclease activity (amount of activity perpolymerase molecule). By normal or naturally associated level is meantthe exonuclease activity of unmodified T7-type polymerase. Normally theassociated activity is about 5,000 units of exonuclease activity per mgof polymerase, measured as described below by a modification of theprocedure of Chase et al. (249 J. Biol. Chem. 4545, 1974). Exonucleasesincrease the fidelity of DNA synthesis by excising any newly synthesizedbases which are incorrectly basepaired to the template. Such associatedexonuclease activities are detrimental to the quality of DNA sequencingreactions. They raise the minimal required concentration of nucleotideprecursors which must be added to the reaction since, when thenucleotide concentration falls, the polymerase activity slows to a ratecomparable with the exonuclease activity, resulting in no net DNAsynthesis, or even degradation of the synthesized DNA.

More importantly, associated exonuclease activity will cause a DNApolymerase to idle at regions in the template with secondary structureimpediments. When a polymerase approaches such a structure its rate ofsynthesis decreases as it struggles to pass. An associated exonucleasewill excise the newly synthesized DNA when the polymerase stalls. As aconsequence numerous cycles of synthesis and excision will occur. Thismay result in the polymerase eventually synthesizing past the hairpin(with no detriment to the quality of the sequencing reaction); or thepolymerase may dissociate from the synthesized strand (resulting in anartifactual band at the same position in all four sequencing reactions);or, a chain terminating agent may be incorporated at a high frequencyand produce a wide variability in the intensity of different fragmentsin a sequencing gel. This happens because the frequency of incorporationof a chain terminating agent at any given site increases with the numberof opportunities the polymerase has to incorporate the chain terminatingnucleotide, and so the DNA polymerase will incorporate achain-terminating agent at a much higher frequency at sites of idlingthan at other sites.

An ideal sequencing reaction will produce bands of uniform intensitythroughout the gel. This is essential for obtaining the optimal exposureof the X-ray film for every radioactive fragment. If there is variableintensity of radioactive bands, then fainter bands have a chance ofgoing undetected. To obtain uniform radioactive intensity of allfragments, the DNA polymerase should spend the same interval of time ateach position on the DNA, showing no preference for either the additionor removal of nucleotides at any given site. This occurs if the DNApolymerase lacks any associated exonuclease, so that it will have onlyone opportunity to incorporate a chain terminating nucleotide at eachposition along the template.

Short primers

The DNA polymerase of the invention is able to utilize primers of 10bases or less, as well as longer ones, most preferably of 4-20 bases.The ability to utilize short primers offers a number of importantadvantages to DNA sequencing. The shorter primers are cheaper to buy andeasier to synthesize than the usual 15-20-mer primers. They also annealfaster to complementary sites on a DNA template, thus making thesequencing reaction faster. Further, the ability to utilize small (e.g.,six or seven base) oligonucleotide primers for DNA sequencing permitsstrategies not otherwise possible for sequencing long DNA fragments. Forexample, a kit containing 80 random hexamers could be generated, none ofwhich are complementary to any sites in the cloning vector.Statistically, one of the 80 hexamer sequences will occur an average ofevery 50 bases along the DNA fragment to be sequenced. The determinationof a sequence of 3000 bases would require only five sequencing cycles.First, a "universal" primer (e.g., Biolabs #1211, sequence 5'GTAAAACGACGGCCAGT 3') would be used to sequence about 600 bases at oneend of the insert. Using the results from this sequencing reaction, anew primer would be picked from the kit homologous to a region near theend of the determined sequence. In the second cycle, the sequence of thenext 600 bases would be determined using this primer. Repetition of thisprocess five times would determine the complete sequence of the 3000bases, without necessitating any subcloning, and without the chemicalsynthesis of any new oligonucleotide primers. The use of such shortprimers is enhanced by including gene 2.5 and 4 protein of T7 in thesequencing reaction.

DNA polymerases of this invention, (i.e., having the above properties)include modified T7-type polymerases. That is the DNA polymeraserequires host thioredoxin as a sub-unit, and they are substantiallyidentical to a modified T7 DNA polymerase or to equivalent enzymesisolated from related phage, such as T3, ΦI, ΦII, H, W31, gh-1, Y, Al122and Sp6. Each of these enzymes can be modified to have propertiessimilar to those of the modified T7 enzyme. It is possible to isolatethe enzyme from phage infected cells directly, but preferably the enzymeis isolated from cells which overproduce it. By substantially identicalis meant that the enzyme may have amino acid substitutions which do notaffect the overall properties of the enzyme. One example of aparticularly desirable amino acid substitution is one in which thenatural enzyme is modified to remove any exonuclease activity. Thismodification may be performed at the genetic or chemical level (seebelow).

Cloning T7 polymerase

As an example of the invention we shall describe the cloning,overproduction, purification, modification and use of T7 DNA polymerase.This enzyme consists of two polypeptides tightly complexed in a one toone stoichiometry. One is the phage T7-encoded gene 5 protein of 84,000daltons (Modrich et al. 150 J. Biol. Chem. 5515, 1975), the other is theE. coli encoded thioredoxin, of 12,000 daltons (Tabor et al., 82 Proc.Natl. Acad. Sci. 1074, 1985). The thioredoxin is an accessory proteinand attaches the gene 5 protein (the actual DNA polymerase) to theprimer template. The natural DNA polymerase has a very active 3' to 5exonuclease associated with it. This activity makes the polymeraseuseless for DNA sequencing and must be inactivated or modified beforethe polymerase can be used. This is readily performed, as describedbelow, either chemically, by local oxidation of the exonuclease domain,or genetically, by modifying the coding region of the polymerase geneencoding this activity.

pTrx-2

In order to clone the trxA (thioredoxin) gene of E. coli wild type E.coli DNA was partially cleaved with Sau3A and the fragments ligated toBamHI-cleaved T7 DNA isolated from strain T7 ST9 (Tabor et al., inThioredoxin and Glutaredoxin Systems: Sturcture and Function (Holmgrenet al., eds) pp. 285-300, Raven Press, NY; and Tabor et al., supra). Theligated DNA was transfected into E. coli trxA⁻ cells, the mixture platedonto trxA⁻ cells, and the resulting T7 plaques picked. Since T7 cannotgrow without an active E. coli trxA gene only those phages containingthe trxA gene could form plaques. The cloned trxA genes were located ona 470 base pair HincII fragment.

In order to overproduce thioredoxin a plasmid, pTrx-2, was asconstructed. Briefly, the 470 base pair HincII fragment containing thetrxA gene was isolated by standard procedure (Maniatis et al., Cloning:A Laboratory Manual, Cold Spring Harbor Labs., Cold Spring Harbor,N.Y.), and ligated to a derivative of pBR322 containing a Ptac promoter(ptac-12, Amann et al., 25 Gene 167, 1983). Referring to FIG. 2,ptac-12, containing β-lactamase and Col El origin, was cut with PvuII,to yield a fragment of 2290 bp, which was then ligated to two tandemcopies of trxA (HincII fragment) using commercially available linkers(SmaI-BamHI Polylinker), to form pTrx-2. The complete nucleotidesequence of pTrx-2 is shown in FIG. 7. Thioredoxin production is nowunder the control of the tac promoter, and thus can be specificallyinduced, e.g. by IPTG (isopropyl β-D-thiogalactoside).

pGP5-5 and mGP1-2

Some gene products of T7 are lethal when expressed in E. coli. Anexpression system was developed to facilitate cloning and expression of,lethal genes, based on the inducible expression of T7 RNA polymerase.Gene 5 protein is lethal in some E. coli strains and an example of sucha system is described by Tabor et al. 82 Proc. Nat. Acad. Sci. 1074(1985) where T7 gene 5 was placed under the control of the Φ10 promoter,and is only expressed when T7 RNA polymerase is present in the cell.

Briefly, pGP5-5 (FIG. 3) was constructed by standard procedures usingsynthetic BamHI linkers to join T7 fragment from 14306 (NdeI) to 16869(AhaIII), containing gene 5, to the 560 bp fragment of T7 from 5667(HincII) to 6166 (Fnu4Hl) containing both the Φ1.1A and Φ1.1B promoters,which are recognized by T7 RNA polymerase, and the 3kb BamHI-HincIIfragment of pACYC177 (Chang et al., 134 J. Bacteriol. 1141, 1978). Thenucleotide sequence of the T7 inserts and linkers in shown in FIG. 8. Inthis plasmid gene 5 is only expressed when T7 RNA polymerase is providedin the cell.

Referring to FIG. 3, T7 RNA polymerase is provided on phage vectormGP1-2. This is similar to pGP1-2 (Tabor et al., id.) except that thefragment of T7 from 3133 (HaeIII) to 5840 (HinfI), containing T7 RNApolymerase was ligated, using linkers (BglII and SalI respectively), toBamHI-SalI cut M13 mp8, placing the polymerase gene under control of thelac promoter. The complete nucleotide sequence of mGP1-2 is shown inFIG. 9.

Since pGP5-5 and pTrx-2 have different origins of replication(respectively a P15A and a ColEl origin) they can be tranformed into onecell simultaneously. pTrx-2 expresses large quantities of thioredoxin inthe presence of IPTG. mGP1-2 can coexist in the same cell as these twoplasmids and be used to regulate expression of T7-DNA polymerase frompGP5-5, simply by causing production of T7-RNA polymerase by inducingthe lac promoter with, e.g., IPTG.

Overproduction of T7 DNA polymerase

There are several potential strategies for overproducing andreconstituting the two gene products of trxA and gene 5. The same cellstrains and plasmids can be utilized for all the strategies. In thepreferred strategy the two genes are co-overexpressed in the same cell.(This is because gene 5 is susceptible to proteases until thioredoxin isbound to it.) As described in detail below, one procedure is to placethe two genes separately on each of two compatible plasmids in the samecell. Alternatively, the two genes could be placed in tandem on the sameplasmid. It is important that the T7-gene 5 is placed under the controlof a non-leaky inducible promoter, such as Φ1.1A, Φ1.1B and Φ10 of T7,as the synthesis of even small quantities of the two polypeptidestogether is toxic in most E. coli cells. By non-leaky is meant that lessthan 500 molecules of the gene product are produced, per cell generationtime, from the gene when the promoter, controlling the gene'sexpression, is not activated. Preferably the T7 RNA polymeraseexpression system is used although other expression systems whichutilize inducible promoters could also be used. A leaky promoter, e.g.,plac, allows more than 500 molecules of protein to be synthesized, evenwhen not induced, thus cells containing lethal genes under the controlof such a promoter grow poorly and are not suitable in this invention.It is of course possible to produce these products in cells where theyare not lethal, for example, the plac promoter is suitable in suchcells.

In a second strategy each gene can be cloned and overexpressedseparately. Using this strategy, the cells containing the individuallyoverproduced polypeptides are combined prior to preparing the extracts,at which point the two polypeptides form an active T7 DNA polymerase.

EXAMPLE 1 Production of T7 DNA polymerase

E. coli strain JM103 (Messing et al., 9 Nuc. Acid Res. 309, 1981) isused for preparing stocks of mGP1-2. JM103 is stored in 50% glycerol at-80° C. and is streaked on a standard minimal media agar plate. A singlecolony is grown overnight in 25 ml standard M9 media at 37° C., and asingle plaque of mGP1-2 is obtained by titering the stock using freshlyprepared JM103 cells. The plaque is used to inoculate 10 ml 2× LB (2%Bacto-Tryptone, 1% yeast extract, 0.5% NaCl, 8 mM NaOH) containing JM103grown to an A₅₉₀ =0.5. This culture will provide the phage stock forpreparing a large culture of mGP1-2. After 3-12 hours, the 10 ml cultureis centrifuged, and the supernatant used to infect the large (2 L)culture. For the large culture, 4×500 ml 2× LB is inoculated with 4×5 ml71.18 cells grown in M9, and is shaken at 37° C. When the large cultureof cells has grown to an A₅₉₀ =1.0 (approximately three hours), they areinoculated with 10 ml of supernatant containing the starter lysate ofmGP1-2. The infected cells are then grown overnight at 37° C. The nextday, the cells are removed by centrifugation, and the supernatant isready to use for induction of K38/pGP5-5/pTrx-2 (see below). Thesupernatant can be stored at 4° C. for approximately six months, at atiter ˜5×10¹¹ Φ/ml. At this titer, 1 L of phage will infect 12 liters ofcells at an A₅₉₀ =5 with a multiplicity of infection of 15. If the titeris low, the mGP1-2 phage can be concentrated from the supernatant bydissolving NaCl (60 gm/liter) and PEG-6000 (65 gm/liter) in thesupernatant, allowing the mixture to settle at 0° C. for 1-72 hours, andthen centrifuging (7000 rpm for 20 min). The precipitate, which containsthe mGP1-2 phage, is resuspended in approximately 1/20th of the originalvolume of M9 media.

K38/pGP5-5/pTrx-2 is the E. coli strain (genotype HfrC (λ)) containingthe two compatible plasmids pGP5-5 and pTrx-2. pGP5-5 plasmid has a P15Aorigin of replication and expresses the kanamycin (Km) resistance gene.pTrx-2 has a ColEI origin of replication and expresses the ampicillin(Ap) resistance gene. The plasmids are introduced into K38 by standardprocedures, selecting Km^(R) and Ap^(R) respectively. The cellsK38/pGP5-5/pTrx-2 are stored in 50% glycerol at -80° C. Prior to usethey are streaked on a plate containing 50 μg/ml ampicillin andkanamycin, grown at 37° C. overnight, and a single colony grown in 10 mlLB media containing 50 μg/ml ampicillin and kanamycin, at 37° C. for 4-6hours. The 10 ml cell culture is used to inoculate 500 ml of LB mediacontaining 50 μ g/ml ampicillin and kanamycin and shaken at 37° C.overnight. The following day, the 500 ml culture is used to inoculate 12liters of 2× LB-KPO₄ media (2% Bacto-Tryptone, 1% yeast extract, 0.5%NaCl, 20 mM KPO₄, 0.2% dextrose, and 0.2% casamino acids, pH 7.4), anaeration in a fermentor at 37° C. When the cells reach an A₅₉₀ =5.0(i.e. logarithmic or stationary phase cells), they are infected withmGP1-2 at a multiplicity of infection of 10, and IPTG is added (finalconcentration 0.5 mM). The IPTG induces production of thioredoxin andthe T7 RNA polymerase in mGP1-2, and thence induces production of thecloned DNA polymerase. The cells are grown for an additional 2.5 hourswith stirring and aeration, and then harvested. The cell pellet isresuspended in 1.5 L 10% sucrose/20 mM Tris-HCl, pH 8.0/25 mM EDTA andre-spun. Finally, the cell pellet is resuspended in 200 ml 10%sucrose/20 mM Tris-HCl, pH 8/1.0 mM EDTA, and frozen in liquid N₂. From12 liters of induced cells 70 gm of cell paste are obtained containingapproximately 700 mg gene 5 protein and 100 mg thioredoxin.

K38/pTrx-2 (K38 containing pTrx-2 alone) overproduces thioredoxin, andit is added as a "booster" to extracts of K38/pGP5-5/pTrx-2 to insurethat thioredoxin is in excess over gene 5 protein at the outset of thepurification. The K38/pTrx-2 cells are stored in 50% glycerol at -80° C.Prior to use they are streaked on a plate containing 50 μg/mlampicillin, grown at 37° C. for 24 hours, and a single colony grown at37° C. overnight in 25 ml LB media containing 50 μg/ml ampicillin. The25 ml culture is used to inoculate 2 L of 2× LB media and shaken at 37°C. When the cells reach an A₅₉₀ =3.0, the ptac promoter, and thusthioredoxin production, is induced by the addition of IPTG (finalconcentration 0.5 mM). The cells are grown with shaking for anadditional 12-16 hours at 37° C., harvested, resuspended in 600 ml 10%sucrose/20 mM Tris-HCl, pH 8.0/25 mM EDTA, and re-spun. Finally, thecells are resuspended in 40 ml 10% sucrose/20 mM Tris-HCl, pH 8/0.5 mMEDTA, and frozen in liquid N₂. From 2L of cells 16 gm of cell paste areobtained containing 150 mg of thioredoxin.

Assays for the polymerase involve the use of single-stranded calf thymusDNA (6 mM) as a substrate. This is prepared immediately prior to use bydenaturation of double-stranded calf thymus DNA with 50 mM NaOH at 20°C. for 15 min., followed by neutralization with HCl. Any purified DNAcan be used as a template for the polymerase assay, although preferablyit will have a length greater than 1,000 bases.

The standard T7 DNA polymerase assay used is a modification of theprocedure described by Grippo et al. (246 J. Biol. Chem. 6867, 1971).The standard reaction mix (200 μl final volume) contains 40 mM Tris/HClpH 7.5, 10 mM MgCl₂, 5 mM dithiothreitol, 100 nmol alkali-denatured calfthymus DNA, 0.3 mM dGTP, dATP, dCTP and [³ H]dTTP (20 cpm/pm), 50 μg/mlBSA, and varying amounts of T7 DNA polymerase. Incubation is at 37° C.(10° C.-45° C.) for 30 min (5 min-60 min). The reaction is stopped bythe addition of 3 ml of cold (0° C.) 1 N HCl-0.1 M pyrophosphate.Acid-insoluble radioactivity is determined by the procedure of Hinkle etal. (250 J. Biol. Chem. 5523, 1974). The DNA is precipitated on ice for15 min (5 min-12 hr), then precipitated onto glass-fiber filters byfiltration. The filters are washed five times with 4 ml of cold (0° C.)0.1 M HCl-0.1 M pyrophosphate, and twice with cold (0° C.) 90% ehanol.After drying, the radioactivity on the filters is counted using anon-aqueous scintillation fluor.

One unit of polymerase activity catalyzes the incorporation of 10 nmolof total nucleotide into an acid-soluble form in 30 min at 37° C., underthe conditions given above. Native T7 DNA polymerase and modified T7 DNApolymerase (see below) have the same specific polymerase activity ±20%,which ranges between 5,000-8,000 units/mg depending upon thepreparation, using the standard assay conditions stated above.

T7 DNA polymerase is purified from he above extracts by precipitationand chromatography techniques. An example of such a purificationfollows.

An extract of frozen cells (200 ml K38/pGP5-5/pTrx-2 and 40 mlK38/pTrx-2) are thawed at 0° C. overnight. The cells are combined, and 5ml of lysozyme (15 mg/ml) and 10 ml of NaCl (5M) are added. After 45 minat 0° C., the cells are placed in a 37° C. water bath until theirtemperature reaches 20° C. The cells are then frozen in liquid N₂. Anadditional 50 ml of NaCl (5M) is added, and the cells are thawed in a37° C. water bath. After thawing, the cells are gently mixed at 0° C.for 60 min. The lysate is centrifuged for one hr at 35,000 rpm in aBeckman 45Ti rotor. The supernatant (250 ml) is fraction I. It containsapproximately 700 mg gene 5 protein and 250 mg of thioredoxin (a 2:1ratio thioredoxin to gene 5 protein).

90 gm of ammonium sulphate is dissolved in fraction I (250 ml) andstirred for 60 min. The suspension is allowed to sit for 60 min, and theresulting precipitate collected by centrifugation at 8000 rpm for 60min. The precipitate is redissolved in 300 ml of 20 mM Tris-HCl pH 7.5/5mM 2-mercaptoethanol/0.1 mM EDTA/10% glycerol (Buffer A). This isfraction II.

A column of Whatman DE52 DEAE (12.6 cm² ×18 cm) is prepared and washedwith Buffer A. Fraction II is dialyzed overnight against two changes of1 L of Buffer A each (the buffer having a final conductivity equal tothat of Buffer A containing 100 mM NaCl). Dialyzed Fraction II isapplied to the column at a flow rate of 100 ml/hr, and washed with 400ml of Buffer A containing 100 mM NaCl. Proteins are eluted with a 3.5 Lgradient from 100 to 400 mM NaCl in Buffer A at a flow rate of 60 ml/hr.Fractions containing T7 DNA polymerase, which elutes at 200 mM NaCl, arepooled. This is fraction III (190 ml).

A column of Whatman P11 phosphocellulose (12.6 cm² ×12 cm) is preparedand washed with 20 mM KPO₄ pH 7.4/5 mM 2-mercaptoethanol/0.1 mM EDTA/10%glycerol (Buffer B). Fraction III is diluted 2-fold (380 ml) with BufferB, then applied to the column at a flow rate of 60 ml/hr, and washedwith 200 ml of Buffer B containing 100 mM KCl. Proteins are eluted witha 1.8 L gradient from 100 to 400 mM KCl in Buffer B at a flow rate of 60ml/hr. Fractions containing T7 DNA polymerase, which elutes at 300 mMKCl, are pooled. This is fraction IV (370 ml).

A column of DEAE-Sephadex A-50 (4.9 cm² ×15 cm) is prepared and washedwith 20 mM Tris-HCl 7.0/0.1 mM dithiothreitol/0.1 mM EDTA/10% glycerol(Buffer C). Fraction IV is dialyzed against two changes of 1 L Buffer Cto a final conductivity equal to that of Buffer C containing 100 mMNaCl. Dialyzed fraction IV is applied to the column at a flow rate of 40ml/hr, and washed with 150 ml of Buffer C containing 100 mM NaCl.Proteins are eluted with a 1 L gradient from 100 to 300 mM NaCl inBuffer C at a flow rate of 40 ml/hr. Fractions containing T7 DNApolymerase, which elutes at 210 mM NaCl, are pooled. This is fraction V(120 ml).

A column of BioRad HTP hydroxylapatite (4.9 cm² ×15 cm) is prepared andwashed with 20 mM KPO₄, pH 7.4/10 mM 2-mercaptoethanol/2 mM Nacitrate/10% glycerol (Buffer D). Fraction V is dialyzed against twochanges of 500 ml Buffer D each. Dialyzed fraction V is applied to thecolumn at a flow rate of 30 ml/hr, and washed with 100 ml of Buffer D.Proteins are eluted with a 900 ml gradient from 0 to 180 mM KPO₄, pH 7.4in Buffer D at a flow rate of 30 ml/hr. Fractions containing T7 DNApolymerase, which elutes at 50 mM KPO₄, are pooled. This is fraction VI(130 ml). It contains 270 mg of homogeneous T7 DNA polymerase.

Fraction VI is dialyzed versus 20 mM KPO₄ pH 7.4/0.1 mMdithiothreitol/0.1 mM EDTA/50% glycerol. This is concentrated fractionVI (˜65 ml, 4 mg/ml), and is stored at -20° C.

The isolated T7 polymerase has exonuclease activity associated with it.As stated above this must be inactivated. An example of inactivation bychemical modification follows.

Concentrated fraction VI is dialyzed overnight against 20 mM KPO₄ pH7.4/0.1 mM dithiothreitol/10% glycerol to remove the EDTA present in thestorage buffer. After dialysis, the concentration is adjusted to 2 mg/mlwith 20 mM KPO₄ pH 7.4/0.1 mM dithiothreitol/10% glycerol, and 30 ml (2mg/ml) aliquots are placed in 50 ml polypropylene tubes. (At 2 mg/ml,the molar concentration of T7 DNA polymerase is 22 μM.)

Dithiothreitol (DTT) and ferrous ammonium sulfate (Fe(NH₄)₂ (SO₄)₂ 6H₂O) are prepared fresh immediately before use, and added to a 30 mlaliquot of T7 DNA polymerase, to concentrations of 5 mM DTT (0.6 ml of a250 mM stock) and 20 μM Fe(NH₄)₂ (SO₄)₂ 6H₂ O (0.6 ml of a 1 mM stock).During modification the molar concentrations of T7 DNA polymerase andiron are each approximately 20 μM, while DTT is in 250× molar excess.

The modification is carried out at 0° C. under a saturated oxygenatmosphere as follows. The reaction mixture is placed on ice within adessicator, the dessicator is purged of air by evacuation andsubsequently filled with 100% oxygen. This cycle is repeated threetimes. The reaction can be performed in air (20% oxygen), but occurs atone third the rate.

The time course of loss of exonuclease activity is shown in FIG. 4. ³H-labeled double-stranded DNA (6 cpm/pmol) was prepared frombacteriophage T7 as described by Richardson (15 J. Molec. Biol. 49,1966). ³ H-labeled single-stranded T7 DNA was prepared immediately priorto use by denaturation of double-stranded ³ H-labeled T7 DNA with 50 mMNaOH at 20° C. for 15 min, followed by neutralization with HCl. Thestandard exonuclease assay used is a modification of the proceduredescribed by Chase et al. (supra). The standard reaction mix (100 μlfinal volume) contained 40 mM Tris/HCl pH 7.5, 10 mM MgCl₂, 10 mMdithiothreitol, 60 nmol ³ H-labeled single-stranded T7 DNA (6 cpm/pm),and varying amounts of T7 DNA polymerase. ³ H-labeled double-stranded TyDNA can also be used as a substrate. Also, any uniformly radioactivelylabeled DNA, single- or double-stranded, can be used for the assay.Also, 3' end labeled single- or double-stranded DNA can be used for theassay. After incubation at 37° C. for 15 min, the reaction is stopped bythe addition of 30 μl of BSA (10 mg/ml) and 25 μl of TCA (100% w/v). Theassay can be run 10° C.-45° C. for 1-60 min. The DNA is precipitated onice for 15 min (1 min-12 hr), then centrifuged at 12,000 g for 30 min (5min-3 hr). 100 μl of the supernatant is used to determine theacid-soluble radioactivity by adding it to 400 μl water and 5 ml ofaqueous scintillation cocktail.

One unit of exonuclease activity catalyzes the acid solubilization of 10nmol of total nucleotide in 30 min under the conditions of the assay.Native T7 DNA polymerase has a specific exonuclease activity of 5000units/mg, using the standard assay conditions stated above. The specificexonulease activity of the modified T7 DNA polymerase depends upon theextent of chemical modification, but ideally is at least 10-100-foldlower than that of native T7 DNA polymerase, or 500 to 50 or lessunits/mg using the standard assay conditions stated above.

Under the conditions outlined, the exonuclease activity decaysexponentially, with a half-life of decay of eight hours. Once per daythe reaction vessel is mixed to distribute the soluble oxygen, otherwisethe reaction will proceed more rapidly at the surface where theconcentration of oxygen is higher. Once per day 2.5 mM DTT (0.3 ml of afresh 250 mM stock to a 30 ml reaction) is added to replenish theoxidized DTT.

After eight hours, the exonuclease activity of T7 DNA polymerase hasbeen reduced 50%, with negligable loss of polymerase activity. The 50%loss is the result of the complete inactivation of exonuclease activityof half the polymerase molecules, rather than a general reduction of therate of exonuclease activity in all the molecules. Thus, after an eighthour reaction all the molecules have normal polymerase activity, halfthe molecules have normal exonuclease activity, while the other halfhave <0.01% of their original exonuclease activity.

When 50% of the molecules are modified (an eight hour reaction), theenzyme is suitable, although suboptimal, for DNA sequencing. For moreoptimum quality of DNA sequencing, the reaction is allowed to proceed togreater than 99% modification (having less than 50 units of exonucleaseactivity), which requires four days.

After four days, the reaction mixture is dialyzed against 2 changes of250 ml of 20 mM KPO. pH 7.4/0.1 mM dithiothreitol/0.1 mM EDTA/50%glycerol to remove the iron. The modified T7 DNA polymerase (˜4 mg/ml)is stored at -20° C.

The reaction mechanism for chemical modification of T7 DNA polymerasedepends upon reactive oxygen species generated by the presence ofreduced transition metals such as Fe² + and oxygen. A possible reactionmechanism for the generation of hydroxyl radicals is outlined below:

    Fe.sup.2+ +O.sub.2 →Fe.sup.3+ +O.sub.2.sup..        (1)

    2 O.sub.2.sup.. +2 H.sup.+ →H.sub.2 O.sub.2 +O.sub.2 (2)

    Fe.sup.2+ +H.sub.2 O.sub.2 →Fe.sup.3+ +OH.sup.. +OH.sup..(3)

In equation 1, oxidation of the reduced metal ion yields superoxideradical, O₂.sup... The superoxide radical can undergo a dismutationreaction, producing hydrogen peroxide (equation 2). Finally, hydrogenperoxide can react with reduced metal ions to form hydroxyl radicals,OH.sup.. (the Fenton reaction, equation 3). The oxidized metal ion isrecycled to the reduced form by reducing agents such as dithiothreitol(DTT).

These reactive oxygen species probably inactivate proteins byirreversibly chemically altering specific amino acid residues. Suchdamage is observed in SDS-PAGE of fragments of gene 5 produced by CNBror trypsin. Some fragments disappear, high molecular weight crosslinking occurs, and some fragments are broken into two smallerfragments.

As previously mentioned, oxygen, a reducing agent (e.g. DTT,2-mercaptoethanol) and a transition metal (e.g. iron) are essentialelements of the modification reaction. The reaction occurs in air, butis stimulated three-fold by use of 100% oxygen. The reaction will occurslowly in the absence of added transition metals due to the presence oftrace quantities of transition metals (1-2 μM) in most bufferpreparations.

As expected, inhibitors of the modification reaction include anaeorobicconditions (e.g., N₂) and metal chelators (e.g. EDTA, citrate,nitrilotriacetate). In addition, the enzymes catalase and superoxidedismutase inhibit the reaction, consistent with the essential role ofreactive oxygen species in the generation of modified T7 DNA polymerase.

As an alternative procedure, it is possible to genetically mutate the T7gene 5 to specifically inactivate the exonuclease domain of the protein.The T7 gene 5 protein purified from such mutants is ideal for use in DNAsequencing without the need to chemically inactivate the exonuclease byoxidation.

Genetically modified T7 DNA polymerase is isolated by randomlymutagenizing the gene 5 and then screening for those mutants that havelost exonuclease activity, without loss of polymerase activity.Mutagenesis is performed as follows. Single-stranded DNA containing gene5 (e.g., cloned in pEMBL-8, a plasmid containing an origin for singlestranded DNA replication) is prepared by standard procedure, and treatedwith two different chemical mutagens: hydrazine, which will mutate C'sand T's, and formic acid, which will mutate G's and A's. Myers et al.229 Science 242, 1985. The DNA is mutagenized at a dose which results inan average of one base being altered per plasmid molecule. Thesingle-stranded mutagenized plasmids are then primed with a universal17-mer primer (see above), and used as templates to synthesize theopposite strands. The synthesized strands contain randomly incorporatedbases at positions corresponding to the mutated bases in the templates.The double-stranded mutagenized DNA is then used to transform the strainK38/pGP1-2, which is strain K38 containing the plasmid pGP1-2 (Tabor etal., supra). Upon heat induction this strain expresses T7 RNApolymerase. The transformed cells are plated at 30° C., withapproximately 200 colonies per plate.

Screening for cells having T7 DNA polymerase lacking exonucleaseactivity is based upon the following finding. The 3' to 5' exonucleaseof DNA polymerases serves a proofreading function. When bases aremisincorporated, the exonuclease will remove the newly incorporated basewhich is recognized as "abnormal". This is the case for the analog ofdATP, etheno-dATP, which is readily incorporated by T7 DNA polymerase inplace of dATP. However, in the presence of the 3' to 5' exonuclease ofT7 DNA polymerase, it is excised as rapidly as it is incorporated,resulting in no net DNA synthesis. As shown in FIG. 6, using thealternating copolymer poly d(AT) as a template, native T7 DNA polymerasecatalyzes extensive DNA synthesis only in the presence of dATP, and notetheno-dATP. In contrast, modified T7 DNA polymerase, because of itslack of an associated exonuclease, stably incorporates etheno-dATP intoDNA at a rate comparable to dATP. Thus, using poly d(AT) as a template,and dTTP and etheno-dATP as precursors, native T7 DNA polymerase isunable to synthesize DNA from this template, while T7 DNA polymerasewhich has lost its exonuclease activity will be able to use thistemplate to synthesize DNA.

The procedure for lysing and screening large number of colonies isdescribed in Raetz (72 Proc. Nat. Acad. Sci. 2274, 1975). Briefly, theK38/pGP1-2 cells transformed with the mutagenized gene 5-containingplasmids are transferred from the petri dish, where they are present atapproximately 200 colonies per plate, to a piece of filter paper("replica plating"). The filter paper discs are then placed at 42° C.for 60 min to induce the T7 RNA polymerase, which in turn expresses thegene 5 protein. Thioredoxin is constitutively produced from thechromosomal gene. Lysozyme is added to the filter paper to lyse thecells. After a freeze thaw step to ensure cell lysis, the filter paperdiscs are incubated with poly d(AT), [α³² P]dTTP and etheno-dATP at 37°C. for 60 min. The filter paper discs are then washed with acid toremove the unincorporated [³² P]dATP. DNA will precipitate on the filterpaper in acid, while nucleotides will be soluble. The washed filterpaper is then used to expose X-ray film. Colonies which have induced anactive T7 DNA polymerase which is deficient in its exonuclease will haveincorporated acid-insoluble ³² P, and will be visible byautoradiography. Colonies expressing native T7 DNA polymerase, orexpressing a T7 DNA polymerase defective in polymerase activity, willnot appear on the autoradiograph.

Colonies which appear positive are recovered from the master petri dishcontaining the original colonies. Cells containing each potentialpositive clone will be induced on a larger scale (one liter) and T7 DNApolymerase purified from each preparation to ascertain the levels ofexonuclease associated with each mutant. Those lacking exonuclease areappropriate for DNA sequencing.

DNA Sequencing Using Modified T7-type DNA Polymerase

DNA synthesis reactions using modified T7-type DNA polymerase result inchain-terminated fragments of uniform radioactive intensity, throughoutthe range of several bases to thousands of bases in length. There isvirtually no background due to terminations at sites independent ofchain terminating agent incorporation (i.e. at pause sites or secondarystructure impediments).

Sequencing reactions using modified T7-type DNA polymerase consist of apulse and chase. By pulse is meant that a short labelled DNA fragment issynthesized; by chase is meant that the short fragment is lengtheneduntil a chain terminating agent is incorporated. The rationale for eachstep differs from conventional DNA sequencing reactions. In the pulse,the reaction is incubated at 0° C.-37° C. for 0.5-4 min in the presenceof high levels of three nucleotide triphosphates (e.g., dGTP, dCTP anddTTP) and limiting levels of one other labelled, carrier-free,nucleotide triphosphate, e.g., [³⁵ S] dATP. Under these conditions themodified polymerase is unable to exhibit its processive character, and apopulation of radioactive fragments will be synthesized ranging in sizefrom a few bases to several hundred bases. The purpose of the pulse isto radioactively label each primer, incorporating maximal radioactivitywhile using minimal levels of radioactive nucleotides. In this example,two conditions in the pulse reaction (low temperature, e.g., from 0°-20°C., and limiting levels of dATP, e.g., from 0.1 μM to 1 μM) prevent themodified T7-type DNA polymerase from exhibiting its processivecharacter. Other essential environmental components of the mixture willhave similar effects, e.g., limiting more than one nucleotidetriphosphate. If the primer is already labelled (e.g., by kinasing) nopulse step is required.

In the chase, the reaction is incubated at 45° C. for 1-30 min in thepresence of high levels (50-500 μM) of all four deoxynucleosidetriphosphates and limiting levels (1-50 μM) of any one of the four chainterminating agents, e.g., dideoxynucleoside triphosphates, such that DNAsynthesis is terminated after an average of 50-600 bases. The purpose ofthe chase is to extend each radioactively labeled primer underconditions of processive DNA synthesis, terminating each extensionexclusively at correct sites in four separate reactions using each ofthe four dideoxynucleoside triphosphates. Two conditions of the chase(high temperature, e.g., from 30°-50° C.) and high levels (above 50 μM)of all four deoxynucleoside triphosphates) allow the modified T7-typeDNA polymerase to exhibit its processive character for tens of thousandsof bases; thus the same polymerase molecule will synthesize from theprimer-template until a dideoxynucleotide is incorporated. At a chasetemperature of 45° C. synthesis occurs at >700 nucleotides/sec. Thus,for sequencing reactions the chase is complete in less than a second.ssb increases processivity, for example, when using dITP, or when usinglow temperatures, or low levels of triphosphates throughout thesequencing reaction.

Either [α³⁵ S]dATP,[α³² P]dATP or fluorescently labelled nucleotides canbe used in the DNA sequencing reactions with modified T7-type DNApolymerase. If an analog is fluorescent then end labelled primers areused, and no probe step is required.

Two components determine the average extensions of the synthesisreactions. First is the length of time of the pulse reaction. Since thepulse is done in the absence of chain terminating agents, the longer thepulse reaction time, the longer the primer extensions. At 0° C. thepolymerase extensions average 10 nucleotides/sec. Second is the ratio ofdeoxynucleoside triphosphates to chain terminating agents in the chasereaction. A modified T7-type DNA polymerase does not discriminateagainst the incorporation of these analogs, thus the average length ofextension in the chase is four times the ratio of the deoxynucleosidetriphosphate concentration to the chain terminating agent concentrationin the chase reaction. Thus, in order to shorten the average size of theextensions, the pulse time is shortened, e.g., to 30 sec. and the ratioof chain terminating agent to deoxynucleoside triphosphate concentrationis raised in the chase reaction. This can be done either by raising theconcentration of the chain terminating agent or lowering theconcentration of deoxynucleoside triphosphate. To lengthen the averagelength of the extensions, the pulse time is increased, e.g., to 3-4 min,and the concentration of chain terminating agent is lowered (e.g., from20 μM to 2 μM) in the chase reaction.

EXAMPLE 2 DNA sequencing using modified T7 DNA polymerase

The following is an example of a sequencing protocol using dideoxynucleotides as terminating agents.

9 μl of single-stranded M13 DNA (mGP1-2, prepared by standardprocedures) at 0.7 mM concentration is mixed with 1 μl of complementarysequencing primer (standard universal 17-mer, 0.5 pmole primer/μl) and2.5 μl 5× annealing buffer (200 mM Tris-HCl, pH 7.5, 50 mM MgCl₂) heatedto 65° C. for 3 min, and slow cooled to room temperature over 30 min. Inthe pulse reaction, 12.5 μl of the above annealed mix was mixed with 1μl dithiothreitol 0.1 M, 2 μl of 3 dNTPs (dGTP, dCTP, dTTP) 3 mM each(P.L Biochemicals, in TE), 2.5 μl [α³⁵ S]dATP, (1500 Ci/mmol, NewEngland Nuclear) and 1 μl of modified T7 DNA polymerase described inExample 1 (0.4 mg/ml, 2500 units/ml, i.e. 0.4 μg, 2.5 units) andincubated at 0° C., for 2 min, after vortexing and centrifuging in amicrofuge for 1 sec. The time of incubation can vary from 30 sec to 20min and temperature can vary from 0° C. to 37° C. Longer times are usedfor sequencing sequences distant from the primer.

4.5 μl aliquots of the above pulse added to each of four tubescontaining the chase mixes, preheated to 45° C. The four tubes, labeledG, A, T, C, each contain trace amounts of either dideoxy (dd) G, A, T,or C (P-L Biochemicals). The specific chase solutions are given below.Each tube contains 1.5 μl dATP 1 mM, 0.5 μl 5× annealing buffer (200 mMTris-HCl, pH 7.5, 50 mM MgCl₂), and 1.0 μl ddNTP 100 μM (where ddNTPcorresponds to ddG,A,T or C in the respective tubes). Each chasereaction is incubated at 45° C. (or 30° C.-50° C.) for 10 min, and then6 μl of stop solution (90% formamide, 10 mM EDTA, 0.1% xylenecyanol)added to each tube, and the tube placed on ice. The chase times can varyfrom 1-30 min.

The sequencing reactions are run on standard, 6% polyacrylamidesequencing gel in 7M urea, at 30 Watts for 6 hours. Prior to running ona gel the reactions are heated to 75° C. for 2 min. The gel is fixed in10% acetic acid, 10% methanol, dried on a gel dryer, and exposed toKodak OMl high-contrast autoradiography film overnight.

EXAMPLE 3 DNA sequencing using limiting concentrations of dNTPs

In this example DNA sequence analysis of mGP1-2 DNA is performed usinglimiting levels of all four deoxyribonucleoside triphosphates in thepulse reaction. This method has a number of advantages over the protocolin example 2. First, the pulse reaction runs to completion, whereas inthe previous protocol it was necessary to interrupt a time course. As aconsequence the reactions are easier to run. Second, with this method itis easier to control the extent of the elongations in the pulse, and sothe efficiency of labeling of sequences near the primer (the first 50bases) is increased approximately 10-fold.

7 μl of 0.75 mM single-stranded M13 DNA (mGP1-2) was mixed with 1 μl ofcomplementary sequencing primer (17-mer, 0.5 pmole primer/μl ) and 2 μl5× annealing buffer (200 mM Tris-HCl pH 7.5, 50 mM MgCl₂, 250 mM NaCl)heated at 65° C. for 2 min, and slowly cooled to room temperature over30 min. In the pulse reaction 10 μl of the above annealed mix was mixedwith 1 μl dithiothreitol 0.1 M, 2 μl of 3 dNTPs (dGTP, dCTP, dTTP) 1.5μM each, 0.5 μl [α³⁵ S]dATP, (1500 Ci/mmol, New England Nuclear) and 2μl modified T7 DNA polymerase (0.1 mg/ml, 2000 units/ml, i.e., 0.2 μg, 2units) and incubated at 37° C. for 5 min. (The temperature and time ofincubation can be varied from 20° C.-45° C. and 1-60 min.,respectively.)

3.5 μl aliquots of the above pulse reaction were added to each of fourtubes containing the chase mixes, which were preheated to 37° C. Thefour tubes, labeled G, A, T, C, each contain trace amounts of eitherdideoxy G, A, T, C. The specific chase solutions are given below. Eachtube contains 0.5 μl 5× annealing buffer (200 mM Tris-HCl pH 7.5, 50 mMMgCl₂, 250 mM NaCl), 1 μl 4dNTPs (dGTP, dATP, dTTP, dCTP) 200 μM each,and 1.0 μl ddNTP 20 μM. Each chase reaction is incubated at 37° C. for 5min (or 20° C.-45° C. and 1-60 min respectively), and then 4 μl of astop solution (95% formamide, 20 mM EDTA, 0.05% xylene-cyanol) added toeach tube, and the tube placed on ice.

EXAMPLE 4 Replacement of dGTP with dITP for DNA sequencing

In order to sequence through regions of compression in DNA, i.e.,regions having compact secondary structure, it is common to use dITP ordeazaguanosine triphosphate (deaza GTP, Mizusawa et al., 14 Nuc. AcidRes. 1319, 1986, and Mills et al., 76 Proc. Natl. Acad. Sci. 2232, 1979)as a replacement for dGTP. We have found that both analogs function wellwith T7-type polymerases, especially with dITP in the presence of ssb.Prferably these reactions are performed with the above describedgenetically modified T7 polymerase, or the chase reaction is for 1-2min., and/or at 20° C. to reduce exonuclease degradation.

Modified T7 DNA polymerase efficiently utilizes dITP or deaza-GTP inplace of dGTP. dITP is substituted for dGTP in both the pulse and chasemixes at a concentration two to five times that at which dGTP is used.In the ddG chase mix ddGTP is still used (not ddITP).

The chase reactions using dITP are sensitive to the residual low levels(about 0.5 units) of exonuclease activity. To avoid this problem, thechase reaction times should not exceed 5 min when dITP is used. It isrecommended that the four dITP reactions be run in conjunction with,rather than to the exclusion of, the four reactions using dGTP. If bothdGTP and dITP are routinely used, the number of required mixes can beminimized by: (1) Leaving dGTP and dITP out of the chase mixes, whichmeans that the four chase mixes can be used for both dGTP and dITP chasereactions. (2) Adding a high concentration of dGTP or dITP (2 μl at 0.5mM and 1-2.5 mM respectively) to the appropriate pulse mix. The twopulse mixes then each contain a low concentration of dCTP,dTTP and [α³⁵S]dATP, and a high concentration of either dGTP or dITP. Thismodification does not adversely effect the quality of the sequencingreactions, and reduces the required number of pulse and chase mixes torun reactions using both dGTP and dITP to six.

The sequencing reaction is as for example 3, except that two of thepulse mixes contain (a) 3 dNTP mix for dGTP: 1.5 μM dCTP,dTTP, and 1 mMdGTP and (b) 3 dNTP mix for dITP: 1.5 μM dCTP,dTTP, and 2 mM dITP. Inthe chase reaction dGTP is removed from the chase mixes (i.e. the chasemixes contain 30 μM dATP,dTTP and dCTP, and one of the fourdideoxynucleotides at 8 μM), and the chase time using dITP does notexceed 5 min.

Deposits

Strains K38/pGP5-5/pTrx-2, K38/pTrx-2 and M13 mGP1-2 have been depositedwith the ATCC and assigned numbers 67,287; 67,286; and 40,303respectively. These deposits were made on Jan. 13, 1987.

Applicants' and their assignees acknowledge their responsibility toreplace these cultures should they die before the end of the term of apatent issued hereon, 5 years after the last request for a culture, or30 years, whichever is the longer, and its responsibility to notify thedepository of the issuance of such a patent, at which time the depositswill be made irrevocably available to the public and all restrictions toaccess to the cultures will be irrevocably removed. Until that time thedeposits will be made available to the Commissioner of Patents under theterms of 37 CFR Section 1-14 and 35 USC Section 112.

OTHER EMBODIMENTS

Other embodiments are within the following claims.

Other uses of the modified DNA polymerases of this invention, which takeadvantage of their processivity, and lack of exonuclease activity,include the direct enzymatic amplification of genomic DNA sequences.This has been described, for other polymerases, by Saiki et al., 230Science 1350, 1985; and Scharf, 233 Science 1076, 1986.

Referring to FIG. 6, enzymatic amplification of a specific DNA regionentails the use of two primers which anneal to opposite strands of adouble stranded DNA sequence in the region of interest, with their 3'ends directed toward one another (see dark arrows). The actual procedureinvolves multiple (10-40, preferably 16-20) cycles of denaturation,annealing, and DNA synthesis. Using this procedure it is possible toamplify a specific region of human genomic DNA over 200,000 times. As aresult the specific gene fragment represents about one part in five,rather than the initial one part in a million. This greatly facilitatesboth the cloning and the direct analysis of genomic DNA. For diagnosticuses, it can speed up the analysis from several weeks to 1-2 days.

Unlike Klenow fragment, where the amplification process is limited tofragments under two hundred bases in length, modified T7-type DNApolymerases should (preferably in conjuction with E. coli DNA bindingprotein, or ssb, to prevent "snapback" formation of double stranded(DNA) permit the amplification of DNA fragments thousands of bases inlength.

The modified T7-type DNA polymerases are also suitable in standardreaction mixtures: for (a) filling in 5' protruding termini of DNAfragments generated by restriction enzyme cleavage; in order to, forexample, produce blunt-ended double stranded DNA from a linear DNAmolecule having a single stranded region with no 3' protruding termini;(b) for labeling the 3' termini of restriction fragments, for mappingmRNA start sites, or sequencing DNA using the Maxam and Gilbert chemicalmodification procedure; and (c) for in vitro mutagenesis of cloned DNAfragments. For example, a chemically synthesized primer which containsspecific mismatched bases is hybridized to a DNA template, and thenextended by the modified T7-type DNA polymerase. In this way themutation becomes permanently incorporated into the synthesized strand.It is advantageous for the polymerase to synthesize from the primerthrough the entire length of the DNA. This is most efficiently doneusing a processive DNA polymerase. Alternatively mutagenesis isperformed by misincorporation during DNA synthesis (see above). Thisapplication is used to mutagenize specific regions of cloned DNAfragments. It is important that the enzyme used lack exonucleaseactivity. By standard reaction mixture is meant a buffered solutioncontaining the polymerase and any necessary deoxynucleosides, or othercompounds.

We claim:
 1. A method for determining the nucleotide base sequence of aDNA molecule, comprising:annealing said DNA molecule with a primermolecule able to hybridize to said DNA molecule; incubating separateportions of the annealed mixture in at least four vessels, each vesselcontaining four different deoxynucleoside triphosphates, a processiveT7-type DNA polymerase, wherein said polymerase remains bound to saidDNA molecule for at least 500 bases before dissociating in anenvironmental condition used in the extension reaction of a DNAsequencing reaction, said polymerse having less than 500 units ofexonuclease activity per mg of said polymerase, and one of four DNAsynthesis terminating agents which terminate DNA synthesis at a specificnucleotide base, wherein each said agent terminates DNA synthesis at adifferent nucleotide base, and separating the DNA products of eachincubating reaction according to their size, whereby at least a part ofthe nucleotide base sequence of said DNA molecule can be determined. 2.The method of claim 1, wherein said polymerase is unable to exhibit itsprocessivity in a second environmental condition normally used in thepulse step of a DNA sequence reaction.
 3. The method of claim 1 whereinsaid polymerase remains bound to said DNA molecule for at least 1,000bases before dissociating.
 4. The method of claim 1 wherein saidpolymerase is that polymerase in cells infected with a T7-type phage. 5.The method of claim 4 wherein said T7-type phage is T7, T3, ΦI, ΦII, H,W31, gh-1, Y, Al122 or Sp6.
 6. The method of claim 1 wherein saidpolymerase is non-discriminating for dideoxy nucleotide analogs.
 7. Themethod of claim 1 wherein said polymerase is a modified polymerasehaving less than 50 units of exonuclease activity per mg of polymerase.8. The method of claim 7 wherein said modified polymerase has less than1 unit of activity per mg of polymerase.
 9. The method of claim 7wherein said modified polymerase has less than 0.1 unit of activity permg of polymerase.
 10. The method of claim 1 wherein said polymerase hasno detectable exonuclease activity.
 11. The method of claim 1 whereinsaid polymerase is able to utilize primers of 10 base pairs or more. 12.The method of claim 1 wherein said polymerase is able to utilize primersof 4 base pairs or more.
 13. The method of claim 1 wherein said primercomprises 4-20 base pairs and said polymerase is able to utilize primersof 4-20 base pairs.
 14. The method of claim 4 wherein said polymerase isnon-discriminating for nucleotide analogs, and is a modified polymerasehaving less than 500 units of exonuclease activity per mg ofpolymerasesaid primer is single stranded RNA or DNA containing 4-10bases and said polymerase is able to utilize primers of 4-10 bases, andsaid incubating comprises a pulse and a chase step.
 15. The method ofclaim 1 wherein said primer is single stranded DNA or RNA.
 16. Themethod of claim 1 wherein said annealing comprises heating said DNAmolecule and said primer to above 65° C., and allowing the heatedmixture to cool to 10° C. to 30° C.
 17. The method of claim 1 whereinsaid incubating comprises a pulse and a chase step.
 18. The method ofclaim 17 wherein said pulse step comprises mixing said annealed mixturewith all four deoxynucleoside triphosphates and a processive DNApolymerase, wherein at least one said deoxynucleoside triphosphate islabelled and present in a limiting concentration.
 19. The method ofclaim 18 wherein said pulse step incubation is carried out for 30seconds to 20 minutes.
 20. The method of claim 18 wherein said chasestep comprises adding one said chain terminating agent to four separatealiquots of the mixture after performing said pulse step.
 21. The methodof claim 20 wherein said chase step incubation is carried out for 1 to60 minutes.
 22. The method of claim 1 wherein said terminating agent isa dideoxynucleotide.
 23. The method of claim 1 wherein said terminatingagent is a limiting level of one deoxynucleoside triphosphate.
 24. Themethod of claim 1 wherein one said deoxynucleoside triphosphate ischosen from dITP or deazaguanosine.
 25. The method of claim 1 whereinsaid primer is labelled prior to said annealing step.
 26. The method ofclaim 25 wherein said incubating comprises a chase step.
 27. The methodof claim 25 wherein said primer is fluorescently labelled.
 28. Themethod of claim 4 wherein said T7-type phage is T7.
 29. The method ofclaim 12 wherein said mixture comprises T7 gene 2.5 or gene
 4. 30. Themethod of claim 13 wherein said mixture comprises T7 gene 2.5 or gene 4.31. A kit for DNA sequencing, comprising:a processive T7-type DNApolymerase, wherein said polymerase remains bound to a DNA molecule forat least 500 bases before dissociating, said polymerase having less than500 units of exonuclease activity per mg of polymerase, said polymerasebeing able to exhibit its processivity in an environmental conditionnormally used in the extension reaction of a DNA sequencing reaction,and a reagent necessary for said sequencing, selected from (a) dITP and(b) a chain terminating agent.
 32. The kit of claim 31 wherein saidpolymerase is unable to exhibit its processivity in a secondenvironmental condition normally used in the pulse step of a DNAsequencing reaction.